University of Groningen
Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds
Zhao, Pengkun
DOI:
10.33612/diss.168542653
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Publication date: 2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Zhao, P. (2021). Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds. University of Groningen. https://doi.org/10.33612/diss.168542653
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Summary
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In the emerging field of polymer mechanochemistry the advantageous properties of ultrasound (US) can be exploited to control properties on a molecular level by rearranging or cleaving bonds site-specifically. For a successful and selective bond activation by mechanical stress, a macromolecular framework is required that transduces the force to the mechanochemically labile bond of the latent molecular motif (the mechanophore). For biomedical applications, the utilization of non-invasive US is particularly beneficial, as it combines spatial and temporal control with easy regulation of the tissue penetration depth by varying frequency and energy through exposure time. While until now research in mechanochemistry mainly focused on understanding the force-induced chemical transformations and their impact on material properties, utilization of site-selective bond-scission for drug activation in the field of medicine remained unexplored. In this context, we came up with two feasible ways to activate drugs and proteins from either poly-nucleic acids or DNA-conjugated nanoparticles by the treatment of ultrasound, aiming to find applications in the biomedical field. Notably, we presented the first molecular design to activate proteins and drugs from their deactivated aptamer-bound parent form by using mechanical force in the form of ultrasound.
In Chapter 1, we highlighted the progress of nucleic acid-based drug delivery systems triggered by external stimuli, including electric field, magnetic field, light and ultrasound. The classification of each stimulus based on different mechanisms were also reviewed by summarizing representative references.
In Chapter 2, we synthesized high-molar-mass poly-aptamers consisting of nucleic acid aptamer repeat units bound to the antibiotics neomycin B (NeoB) or paromomycin (Paromo). Application of US destroyed the non-covalent interactions (such as hydrogen bonds or electrostatic interactions), led to additional disaggregation and covalent bond scission of the phosphodiester RNA backbone, and subsequently activated the drugs. The minimal inhibitory concentration (MIC) tests of NeoB@poly-aptamers decreased to 8 μg/ml after 10 min and reached the MIC of pristine NeoB after 30 min of ultrasonication. Importantly, small-molecule NeoB@aptamers did not show this behaviour, indicating that these results originate from the mechanochemical scission of non-covalent host-guest interactions and covalent degradation of the nucleic acid backbone. Live/dead staining assay of Staphylococcus aureus (S.aureus) further
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demonstrated that while pristine NeoB killed almost all bacteria, NeoB@poly-aptamers showed activity only after ultrasonication, but not before. This work expands the usage of mechanochemistry to the pharmaceutical sector.
In the same context, we further explored the protein thrombin as payload for the nucleic acid scaffolds to achieve ultrasound-controlled activation in Chapter 3. The proteins were encapsulated and temporally deactivated by poly-aptamers, which were fabricated by rolling circle amplification (RCA). Again, ultrasound was exploited as a non-invasive and deeply penetrating trigger to activate enzyme activity. After 30-60 seconds US application, near-complete recovery of the original thrombin catalytic activity (80-90%) for fibrin formation was observed. Optical microscopy experiments further proved that thrombin@poly-aptamers treated with US for 30-60 s resulted in dense fibrin fiber networks similar to pristine thrombin. Longer US exposition reduced the activity, as thrombin itself is a biomacromolecule which denatures under shear force in solution. Proteins activated by applying US in a molecularly precise fashion was demonstrated in this work.
In addition to the biomacromolecule scaffolds discussed in Chapter 2 and Chapter 3, the focus was shifted to a nanoparticle-based platform for protein activation in Chapter
4. Here, we prepared an aptamer-nanoparticle system to deactivate and subsequently
release thrombin. Therefore, we prepared two types of gold nanoparticles (AuNPs) functionalized on the surfaces with a thiolated split aptamer. In the presence of thrombin, the two split aptamer parts assembled into the intact aptamer tertiary structure and at the same time induced the aggregation of the two AuNP types. With the treatment of ultrasound, these aggregates disassembled, and thrombin was released and hence activated. Already 15 s of US irradiation resulted in 50% of pristine thrombin activity and more than 70% activity was obtained after 30 s. Notably, this process could be manipulated for several cycles without obvious fatigue rendering this hybrid structure reversible. The sample was transformed to its disassembled state simply by US irradiation for 15 s and returned to its assembled state by equilibrating at room temperature for 30 min, as indicated by the SPR band in UV/Vis measurements. This approach circumvents the limitations of single mechanophore architectures suffering from low loading ratios to release sufficient amounts of functional species or the difficulty to accurately control the chain-centered position of the mechanophore.
Summary
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In Chapter 5, we developed a rapid and simple method to differentiate between association and disassociation of proteins and nucleic acids inspired by work on AuNPs with positive charges, which have been successfully tested in detecting antibiotic residues in raw milk. The electrostatic interaction between positively charged AuNPs and polyanionic DNA (lysozyme binding aptamer (LBA)) leads to the aggregation of cationic AuNPs accompanied by a rapid red-to-blue color change. Negative charges of LBA would be diluted with the binding of lysozyme (Lys) as a ratio of 1:1, resulting in the remaining of the red color of individual AuNPs. If the Lys-LBA complex is challenged by employing ultrasound for 1 min, disassociation takes place and AuNPs again could interact with LBA electrostatically. In this case, the aggregation of AuNPs would take place and the red-to-blue color change would be observed again. The most important feature of this assay is direct visualization of the interactions of proteins with nucleic acids by the naked eye, which makes it more convenient than other methods that rely on advanced instruments and laborious characterization procedures.
